Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.9e10229

Porcine pulmonary valve decellularization with NaOH-based vs detergent process: preliminary in vitro and in vivo assessments.

Glutaraldehyde fixed xenogeneic heart valve prosthesis are hindered by calcification and lack of growth potential. The aim of tissue decellularization is to remove tissue antigenicity, avoiding the use of glutaraldehyde and improve valve integration with low inflammation and host cell recolonization. In this preliminary study, we investigated the efficacy of a NaOH-based process for decellularization and biocompatibility improvement of porcine pulmonary heart valves in comparison to a detergent-based process (SDS-SDC0, 5%). Native cryopreserved porcine pulmonary heart valves were treated with detergent and NaOH-based processes. Decellularization was assessed by Hematoxylin and eosin/DAPI/alpha-gal/SLA-I staining and DNA quantification of native and processed leaflets, walls and muscles. Elongation stress test investigated mechanical integrity of leaflets and walls (n = 3 tests/valve component) of valves in the native and treated groups (n = 4/group). Biochemical integrity (collagen/elastin/glycosaminoglycans content) of leaflet-wall and muscle of the valves (n = 4/group) was assessed and compared between groups with trichrome staining (Sirius Red/Miller/Alcian blue). Secondly, a preliminary in vivo study assessed biocompatibility (CD3 and CD68 immunostaining) and remodeling (Hematoxylin and eosin/CD31 and ASMA immunofluorescent staining) of NaOH processed valves implanted in orthotopic position in young Landrace pigs, at 1 (n = 1) and 3 months (n = 2). Decellularization was better achieved with the NaOH-based process (92% vs 69% DNA reduction in the wall). Both treatments did not significantly alter mechanical properties. The detergent-based process induced a significant loss of glycosaminoglycans (p < 0,05). In vivo, explanted valves exhibited normal morphology without any sign of graft dilatation, degeneration or rejection. Low inflammation was noticed at one and three months follow-up (1,8 +/- 3,03 and 0,9836 +/- 1,3605 CD3 cells/0,12 mm <sup>2</sup> in the leaflets). In one animal, at three months we documented minimal calcification in the area of sinus leaflet and in one, microthrombi formation on the leaflet surface at 1 month. The endoluminal side of the valves showed partial reendothelialization. NaOH-based process offers better porcine pulmonary valve decellularization than the detergent process. In vivo, the NaOH processed valves showed low inflammatory response at 3 months and partial recellularization. Regarding additional property of securing, this treatment should be considered for the new generation of heart valves prosthesis. Graphical abstract of the study

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body

Morphology of bile salts micelles and mixed micelles with lipolysis products, from scattering techniques and atomistic simulations

Hypotheses Bile salts (BS) are biosurfactants released into the small intestine, which play key and contrasting roles in lipid digestion: they adsorb at interfaces and promote the adsorption of digestive enzymes onto fat droplets, while they also remove lipolysis products from that interface, solubilising them into mixed micelles. Small architectural variations on their chemical structure, specifically their bile acid moiety, are hypothesised to underlie these conflicting functionalities, which should be reflected in different aggregation and solubilisation behaviour. Experiments The micellisation of two BS, sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC), which differ by one hydroxyl group on the bile acid moiety, was assessed by pyrene fluorescence spectroscopy, and the morphology of aggregates formed in the absence and presence of fatty acids (FA) and monoacylglycerols (MAG) – typical lipolysis products – was resolved by small-angle X-ray/neutron scattering (SAXS, SANS) and molecular dynamics simulations. The solubilisation by BS of triacylglycerol-incorporating liposomes – mimicking ingested lipids – was studied by neutron reflectometry and SANS. Findings Our results demonstrate that BS micelles exhibit an ellipsoidal shape. NaTDC displays a lower critical micellar concentration and forms larger and more spherical aggregates than NaTC. Similar observations were made for BS micelles mixed with FA and MAG. Structural studies with liposomes show that the addition of BS induces their solubilisation into mixed micelles, with NaTDC displaying a higher solubilising capacity

Structure and conformation of wild-type bacterial lipopolysaccharide monolayers at air/water interfaces

The outer membrane of Gram-negative bacteria is of great scientific interest because it mediates the action of antimicrobial agents. The membrane surface is composed of lipopolysaccharide (LPS) molecules with negatively charged oligosaccharide headgroups. To a certain fraction, LPSs additionally display linear polysaccharides termed O-side chains (OSCs). Structural studies on bacterial outer surfaces models, based on LPS monolayers at air-water interfaces, have so far dealt only with rough mutant LPSs lacking these OSCs. Here, we characterize monolayers of wild-type LPS from Escherichia coli O55:B5 featuring strain-specific OSCs in the presence of defined concentrations of monovalent and divalent ions. Pressure-area isotherms yield insight into in-plane molecular interactions and monolayer elastic moduli. Structural investigations by x-ray and neutron reflectometry reveal the saccharide conformation and allow quantifying the area per molecule and the fraction of LPS molecules carrying OSCs. The OSC conformation is satisfactorily described by the self-consistent field theory for end-grafted polymer brushes. The monolayers exhibit a significant structural response to divalent cations, which goes beyond generic electrostatic screening

Deciphering lipid transfer between and within membranes with time-resolved small-angle neutron scattering

This review focuses on time-resolved neutron scattering, particularly time-resolved small angle neutron scattering (TR-SANS), as a powerful in situ noninvasive technique to investigate intra- and intermembrane transport and distribution of lipids and sterols in lipid membranes. In contrast to using molecular analogues with potentially large chemical tags that can significantly alter transport properties, small angle neutron scattering relies on the relative amounts of the two most abundant isotope forms of hydrogen: protium and deuterium to detect complex membrane architectures and transport processes unambiguously. This review discusses advances in our understanding of the mechanisms that sustain lipid asymmetry in membranes—a key feature of the plasma membrane of cells—as well as the transport of lipids between membranes, which is an essential metabolic process

Le Monde

05 juillet 18711871/07/05 (N55,A12).Appartient à l’ensemble documentaire : BbLevt

Spontaneous formation of cushioned model membranes promoted by an intrinsically disordered protein

In this article, it is shown that by exposing commonly used lipids for biomembrane mimicking studies, to a solution containing the histidine-rich intrinsically disordered protein histatin 5, a protein cushion spontaneously forms underneath the bilayer. The underlying mechanism is attributed to have an electrostatic origin, and it is hypothesized that the observed behavior is due to proton charge fluctuations promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Hence, we anticipate that this novel “green” approach of forming cushioned bilayers can be an important tool to mimic the cell membrane without the disturbance of the solid substrate, thereby achieving a further understanding of protein–cell interactions

Flexibility and drug release features of lipid/saccharide nanoparticles

The effect of lipophilic additives (excipients and drugs) on the behavior of lipid/saccharide nanoparticles has been investigated by incoherent elastic neutron scattering. Temperature scans from 20 K to 350 K have been performed on lecithin/chitosan particles loaded with isopropyl myristate and cetyl-stearyl alcohol, two lipophilic molecules with different melting temperatures which are commonly added to improve drug loading efficiency. In a similar way the effect of tamoxifen citrate, a lipophilic drug frequently used in breast cancer therapy, has also been studied. The different melting points of the two excipients affect mostly the low-temperature behavior of the nanoparticles. At physiological temperatures they both improve the particle flexibility. On the other hand addition of tamoxifen leads to stiffer structures and to lower amounts of released drug. The macroscopic features of the drug release appear to be correlated to the microscopic flexibility determined by neutron scattering. The data confirm also the role of chitosan as a stiffening and stabilizing agent of the lipid particles. © 2010 The Royal Society of Chemistry

FT-IR spectral profiles after peak fitting for the liver DNA from NOD mice.

<p>Normoglycemic mice, A; hyperglycemic mice, B. Spectral range: 2990–2850 cm⁻¹. Software: Grams/AI 8.0; function: Gaussian; sensitivity: low.</p